Osteocytes arise from osteoblasts that become surrounded by bone matrix and form a junctionally-connected sensory network that transduces physical strain into signals that maintain bone mass and skeletal integrity. Osteocytes are characterized by membrane extensions (dendrites) with which these cells communicate with neighboring osteocytes and osteoblasts. A loss of osteocyte connectivity is associated with pathological conditions such as osteopenia and osteoarthritis. The proteins responsible for the formation and function of osteocyte dendrites are unknown, and the current lack of information regarding the relationship between osteocyte morphology and function represents a critical gap in the understanding of bone physiology. This project is designed to identify proteins and post-translational processing events that are coupled to osteocyte dendrite outgrowth. The lipid growth factor lysophosphatidic acid (LPA) has pleiotropic effects on bone cells, and recent data revealed that this lipid is a potent inducer of osteocyte dendrite outgrowth in vitro. Another goal of this study is to identify potential mechanisms that are responsible for LPA-induced dendritogenesis. In the first Aim, technological innovations are proposed to identify the complete protein composition (proteome) of mouse osteocytes, and to identify proteins that become enriched in dendrites in response to LPA treatment. A novel method is proposed for the physical separation of osteocyte dendrites from cell bodies using MLO-Y4 osteocytes grown on microporous membranes. These samples will be subjected to state-of-the-art mass spectrometry-based proteomic analysis using analytical capabilities that are unique to the PI's institution. The distribution of dendrite-associated proteins will be verified using western blot and immunofluorescence microscopy. We expect to identify proteins that have potential roles in dendrite outgrowth, as well other functions such as mechanotransduction. Aim 2 is designed to test the hypothesis that LPA induces dendrite formation in osteocytes via a receptor-linked mechanism that requires protein phosphorylation. The experimental approach will include the unprecedented application of mass spectrometry methodology to reveal the phosphoproteome of LPA-treated osteocyte cell fractions. Proteomic and phosphoproteomic data will be subjected to advanced bioinformatics tools to identify signaling pathways that have potential links to dendrite outgrowth. Experiments also include transwell assays for the quantitation of MLO-Y4 cell dendritogenesis, and the inclusion of protein kinase inhibitors to verify the role of specific phosphorylation events in LPA-induced dendrite outgrowth. These studies will provide valuable new insight into the structure and function of osteocytes and contribute to the formulation of mechanistic models of osteocyte dendrite formation. The elucidation of the LPA-linked proteome/phosphoproteome of osteocyte dendrites may provide unique targets for the future treatment of bone wasting diseases and disuse atrophies. PUBLIC HEALTH RELEVANCE: Osteocytes are bone cells that form a network responsible for transducing physical strain into signals that maintain the mass and mechanical strength of the skeleton. This project is designed to identify proteins and regulatory events that control the structure and function of osteocytes. The results of this study will further our understanding of bone cell function, and may provide valuable targets for future drugs as countermeasures to pathological conditions such as osteoarthritis or bone loss due to extended bed rest or long-term exposure to microgravity.